Name: huvec tube-formation assay to evaluate the impact of natural products on angiogenesis
Uploaded: 2019-06-24T13:00:00
Description: Watch this Scientific Journal Video about HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis at JoVE.com

这项工作由方迪·迪阿泰奥资助卢卡·科卢奇-达马托,VALERE计划资金资助玛丽亚·特雷莎·詹蒂尔,AIRC基金IG18999向毛里齐奥·比富尔科提供。

1. RGWE 准备
<ol><li>在植物学家的监督下,在春季/夏季从植物中收集R. 坟墓叶。 注:在这种情况下,叶子被收集在意大利那不勒斯植物园的药用植物实验科5。该植物是自发的,多年生,并存在于地中海地区(图1)。</li>
	<li>称量250克叶子,用剪刀切碎。</li>
	<li>将叶子放入圆锥形烧瓶中,将1升蒸馏水加入250克切碎的叶子中,在110°C下煮沸60分钟。用?...

<ol>
	<li>Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+life,+disease+and+medicine.">Angiogenesis in life, disease and medicine.</a> Nature. 438 (7070), 932-936 (2005).</li><li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+clinical+applications+of+angiogenesis.">Molecular mechanisms and clinical applications of angiogenesis.</a> Nature. 473 (7347), 298-307 (2011).</li><li>Ferrara, N., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+as+a+therapeutic+target.">Angiogenesis as a therapeutic target.</a> Nature. 438 (7070), 967-974 (2005).</li><li>Ravishankar, D., Rajora, A. K., Greco, F., Osborn, H. M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavonoids+as+prospective+compounds+for+anti-cancer+therapy.">Flavonoids as prospective compounds for anti-cancer therapy.</a> The International Journal of Biochemistry &amp; Cell Biology. 45, 2821-2831 (2013).</li><li>Gentile, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ruta+graveolens+water+extract+inhibits+cell-cell+network+formation+in+human+umbilical+endothelial+cells+via+MEK-ERK1/2+pathway.">Ruta graveolens water extract inhibits cell-cell network formation in human umbilical endothelial cells via MEK-ERK1/2 pathway.</a> Experimental Cell Research. 364 (1), 50-58 (2018).</li><li>Butler, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+products+to+drugs:+natural+product-derived+compounds+in+clinical+trials.">Natural products to drugs: natural product-derived compounds in clinical trials.</a> Natural Product Reports. 25, 475-516 (2008).</li><li>Sulaiman, R. S., Basavarajappa, H. D., Corson, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+product+inhibitors+of+ocular+angiogenesis.">Natural product inhibitors of ocular angiogenesis.</a> Experimental Eye Research. 129, 161-171 (2014).</li><li>Risau, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+angiogenesis.">Mechanisms of angiogenesis.</a> Nature. 386 (6626), 671-674 (1997).</li><li>Trung, N. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+for+angiogenesis.">In vitro models for angiogenesis.</a> Journal of Science &amp; Development. 13 (4), 850-858 (2015).</li><li>Ucuzian, A. A., Greisler, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Models+of+Angiogenesis.">In vitro Models of Angiogenesis.</a> World Journal of Surgery. 31, 654-663 (2007).</li><li>Simons, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Heart+Association+Council+on+Basic+Cardiovascular+Sciences+and+Council+on+Cardiovascular+Surgery+and+Anaesthesia.+State-of-the-Art+Methods+for+Evaluation+of+Angiogenesis+and+Tissue+Vascularisation:+A+Scientific+Statement+From+the+American+Heart+Association.">American Heart Association Council on Basic Cardiovascular Sciences and Council on Cardiovascular Surgery and Anaesthesia. State-of-the-Art Methods for Evaluation of Angiogenesis and Tissue Vascularisation: A Scientific Statement From the American Heart Association.</a> Circulation Research. 116 (11), e99-e132 (2015).</li><li>Arnaoutova, I., Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+angiogenesis:+endothelial+cell+tube+formation+on+gelled+basement+membrane+extract.">In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract.</a> Nature Protocols. 5 (4), 628-635 (2010).</li><li>DeCicco-Skinner, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+tube+formation+assay+for+the+in+vitro+study+of+angiogenesis.">Endothelial cell tube formation assay for the in vitro study of angiogenesis.</a> Journal of Visualized Experiments. (91), e51312 (2014).</li></ol>

天然化合物具有高化学多样性和生化特异性,是潜在治疗性分子的来源。在这里,我们展示如何从植物R.重兆中提取水提取物,并提出管形测定作为一种易于执行、可靠和定量的方法,可用于研究RGWE对血管生成的影响。务必将R.黄叶煮沸1小时,以确保获得完整的水萃取物。沸腾不到1小时不允许提取所有分子,提取物不能发挥预期的生物效应。
管形成测定代表一种体外测?...

血管生成是一个生理过程,导致从先前存在的血管形成新的血管,并在胚胎生成和器官生长期间发生。在成年后,血管生成仅在循环卵巢、妊娠期间胎盘以及伤口愈合和修复期间激活。血管生成取决于内皮细胞增殖、分化和迁移的能力,从而形成完整的血管网络1。然而,在几种疾病,如炎症,代谢和风湿病,血管生成过程被改变和血管生成变得过度。此外,不受控制的血管生成过程也刺激肿瘤进展和转移1。由于这些原因,在过去十年中,研究的重点是开发新的治疗策略,旨在抑制癌症,眼,关节,或皮肤疾病的过度血管生成2,3。
血管内皮生长因子(VEGF)是当前抗血管生成疗法4的主要目标,并开发和合成了几种抗VEGF单克隆抗体,以防止血管生成过度。然而,这些合成药物表现出严重的副作用,并且有不利的成本效益比5,6。因此,必须找到新的治疗策略,以限制过度血管生成与最小的副作用,以补充和结合目前使用的药物。这些新药可以在具有高化学多样性和生化特异性的天然产品中找到。
本文提出了一种评估RGWE对HUVEC在体外凝胶基底基质上形成小管能力影响的简单方法。事实上,RGWE是亚次代谢物的混合物,如黄酮类化合物和多酚,其中鲁丁是主要成分5。他们中的许多人已经测试为抗炎和血管保护剂7,8,9,10,11。此外,我们最近已经证明,RGWE,但不是鲁丁,能够抑制HUVEC的能力,形成细胞在凝胶基底基质,这种现象是由MEK-ERK通路,表明RGWE作为一个潜在的治疗工具,能够防止过度的新血管形成5。

<table>
	<tbody>
		<tr>
			<td>HUVEC cells</td>
			<td>Clontech</td>
			<td>C2519A</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Invitrogen</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM-2 basal medium</td>
			<td>Clontech</td>
			<td>cc3156</td>
			<td></td>
		</tr>
		<tr>
			<td>Single quot kit- supplemets and growth factors</td>
			<td>clontech</td>
			<td>cc4147</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Thermo Scientific</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Sarstedt</td>
			<td>62,554,502</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL disposable serological pipette</td>
			<td>Sarstedt</td>
			<td>861,254,001</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL disposable serological pipette</td>
			<td>Sarstedt</td>
			<td>861,253,001</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L pipette</td>
			<td>Gilson</td>
			<td>Pipetman classic</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;L pipette</td>
			<td>Gilson</td>
			<td>Pipetman classic</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L pipette</td>
			<td>Gilson</td>
			<td>Pipetman classic</td>
			<td></td>
		</tr>
		<tr>
			<td>p1000 pipette tips</td>
			<td>Sarstedt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p20-200 pipette tips</td>
			<td>Sarstedt</td>
			<td>70,760,502</td>
			<td></td>
		</tr>
		<tr>
			<td>Burker chamber</td>
			<td>Fortuna</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blu stain</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>mill-ex 0.22 um filters</td>
			<td>Millipore</td>
			<td>SLGS033SS</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>VirTis-SP Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2</td>
			<td>AirCos</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm dish</td>
			<td>Sarstedt</td>
			<td>833,902</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3</td>
			<td>Invitrogen</td>
			<td>v79020</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine-2000</td>
			<td>Invitrogen</td>
			<td>11668027</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td>Reduced serum medium</td>
		</tr>
		<tr>
			<td>Rutin</td>
			<td>Sigma-Aldrich</td>
			<td>R5143-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovert 25 microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AmScope MD500 camera</td>
			<td>AmScope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Thermo Scientific</td>
			<td>D4818</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab heater</td>
			<td>Falc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ParaFilm</td>
			<td>American National Can</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

huvec tube-formation assay to evaluate the impact of natural products on angiogenesis

血管生成是一种包括不同过程的现象,如内皮细胞增殖、分化和迁移,导致新血管的形成,并涉及多个信号转导途径。这里我们表明,管形成测定是一种简单的体外方法,用于评估天然产物对血管生成的影响,并研究所涉及的分子机制。特别是,在Ruta真分子(RGWE)的水提取物存在的情况下,内皮细胞不再能够形成细胞网络,RGWE对人类脐带静脉内皮细胞(HUVEC)管的形成的影响被废除MEK的构成激活。

为了评估RGWE对血管生成的影响,我们对凝胶基底基体进行了管形成测定。在它上培养时,HUVECs形成类似管的结构,这些结构来自看似拉长的细胞,并相互连接,形成细胞网络(图2)。在图3中,我们发现使用RGWE处理的HUVEC中的分支数量与控制条件相比要低得多。值得注意的是,在0.1毫克/mL和1毫克/mL RGWE的情况下,与对照条件相比,分支的数量?...

Watch this Scientific Journal Video about HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis at JoVE.com

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

HUVEC 管形成测定评估天然产物对血管生成的影响

本文利用凝胶基底基体上的管形测定,评估鲁塔墓构的水萃取物对血管网络形成的影响。

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

管形成分析是一种体外测试，用于研究导致形成新血管的几个步骤的分子机制。这种测定允许识别与血管生成相关的化合物和信号级联。通过一个测试，我们能够证明像RGWE这样的天然化合物会降低内皮细胞形成管状结构的能力，不会影响细胞的生存能力，而且这种效果取决于 MEK/ERK 通路的激活。使用正确的单元格数执行测试非常重要。事实上，太少或太多的细胞不能允许正确的形成管。因此，建议进行初步测试，以找出正确的细胞数。卢卡·奥尔加·帕斯托里诺，我实验室的博士生，将展示程序。首先，在春季和夏季收集鲁塔墓碑叶。将250克切碎的叶子放入圆锥形烧瓶中，加入一升蒸馏水、煮沸和过滤，如手稿中所述。第二天，使用冻干剂对叶提取物进行冻干。获得粉末后，称重，并将其分成等分。根据文本协议，在内皮细胞生长介质中培养HUVEC。当细胞达到80%的汇合时，以一比三的比例通过细胞。使用第二通道和第五通道之间获得的细胞。一旦通过的HUVECs达到70%的汇合，用含有感兴趣的基因的载体的一微克将其转接。 将一微克DNA与稀释的利波卡明2000的三微升混合。然后，从HUVECs中去除该介质，用一毫升新鲜完整介质代替。将转式溶液加入细胞中，在37摄氏度和5%的二氧化碳下孵育细胞。转经3小时后，加入7毫升新鲜完整介质，再孵育细胞24至48小时。在地下室准备之前，在冰上放一个预冷却的96井板，并在每一个井中加入50微升的冷基质。当板孵育时，用一毫升的PBS/EDTA溶液清洗HUVEC。然后，向细胞中加入一毫升的 Trypsin-EDTA 溶液，在37摄氏度下孵育5分钟。然后，将细胞重新在一毫升PBS中重新发送。将 0.2 毫升的细胞悬浮液转移到 0.5 毫升的 PBS 中，并使用 trypan 蓝色溶液。在室温下保持悬浮液五分钟后，在 B 中计算细胞数

Research

JoVE Journal

Developmental Biology

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

科研

发育生物学

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

的发展<em&gt;体外</em&gt;检测以确定间充质细胞将经历上皮 - 间质转化的收缩功能

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

竞争性移植评价造血干细胞健身

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

斑马鱼高血糖的急性和慢性模型：评估高血糖对神经发生的影响和放射性标记的分子的生物分布的方法

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

5-mC点印迹测定定量软骨细胞去分化的DNA甲基化水平<em&gt;体外</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

天然铀对体外破影响的分析方法

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

血管血管和血管生成的动力学分析体外

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

体外检测评估神仙体头三个月滋养细胞细胞系的迁移、入侵和增殖

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...